Macrophage (Mphi) death is an important feature of atherosclerotic lesions, yet there are still uncertainties about the physiological consequences of this event. Our laboratory has shown that free cholesterol (FC) loading of cultured can be prevented by FC-induced up-regulation of phosphatidylcholine (PC) biosynthesis and by blockage of FC transport to peripheral cellular sites (e.g., plasma membrane & mitochondria). In this context, the overall objective of this proposal is to test specific hypotheses regarding Mphi death in atherosclerosis using mice in which lipid metabolic and death-signaling pathways have been genetically manipulated. The hypotheses are that Mphi may (a) be protective by limiting the number of Mphi's during lesion development and perhaps by "safely" disposing of dying Mphi's in advanced lesions; and/or (b) contribute to lipid core development by promoting the release of harmful molecules from dying Mphi's. Protective effects may occur when Mphi's die by "apoptosis", whereas harmful effects may occur when Mphi's die by more "necrotic"-like processes. In Aim I, we will use apolipoprotein E knockout (atherosclerotic) mice with Mphi-specific alterations in PC biosynthesis (via; genetic manipulation of CTP: phosphocholine cytidylylyltransferase) or with defective FC transport to peripheral cellular sites (Niemann-Pick C mice). The goal will be to test the role of PC and FC metabolism in Mphi death in vivo and to evaluate, in the context of our hypotheses, the consequences of altered Mphi death on atherogenesis and lipid core development. In Aim II, we will specifically examine mouse models in which Mphi apoptosis should be blocked. In view of preliminary data showing the FC-mediated apoptosis is prevented in Mphi's lacking the Fas death receptor, a major focus will be on mice in which Fas is absent in Mphi's. The role of the Fas pathway in Mphi death caused by older inducers, such as oxidized lipoproteins and growth factor withdrawal, will also be explored. Furthermore, given the key role of bcl- 2 family proteins in certain types of Fas-mediated apoptosis as well as in death due to other causes, mice whose Mphi's over-express the anti- apoptotic proteins Bcl-2 and Bcl-xL will be examined for atherogenesis and lipid core development. Blockage of lesional Mphi apoptosis may, according the above-state hypothesis, adversely affect atherogenesis in necrotic-like changes are left uninhibited. In summary, this project should help elucidate lipid-based mechanisms and physiologic consequences of Mphi death in atherogenesis and lipid core development.